1521-0111/93/5/504–514$35.00 https://doi.org/10.1124/mol.117.111518 MOLECULAR PHARMACOLOGY Mol Pharmacol 93:504–514, May 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics “Selective” Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu 5 Allosteric Ligands s Shane D. Hellyer, Sabine Albold, Taide Wang, Amy N. Y. Chen, Lauren T. May, Katie Leach, and Karen J. Gregory Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia Received December 20, 2017; accepted March 1, 2018 ABSTRACT Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and thera- peutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assess- ment. Using mGlu 5 as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu 5 but exhibited neutral cooperativity with mGlu 5 agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa 21 ) mobilization, and inositol monophosphate (IP 1 ) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu 5 (HEK293A-mGlu 5 -low) for diverse allosteric chemotypes. Numerous “non-mGlu 5 ” class C GPCR allosteric modulators incompletely displaced allosteric mGlu 5 radioligand [ 3 H]- methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu 1 ligand), PHCCC (mGlu 4 ligand), GS39783 (GABA B ligand), AZ12216052 (mGlu 8 ligand), and CGP7930 (GABA B ligand) at mGlu 5 were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu 5 agonists in functional as- says; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu 5 agonists for IP 1 accumulation, but not iCa 21 mobilization. By using mGlu 5 as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinter- preted owing to unappreciated bias. Introduction Class C G protein–coupled receptors (GPCRs) comprise the metabotropic glutamate receptors (mGlu 1 through mGlu 8 ), calcium-sensing receptor (CaSR), GPRC6A, g-aminobutyric acid receptor B (GABA B ), and numerous taste, pheromone, and orphan receptors (Leach and Gregory, 2017). Given the important physiologic and pathophysiologic roles that class C GPCRs play and their putative therapeutic potential, exten- sive drug discovery efforts have been undertaken (Conn et al., 2014; Leach and Gregory, 2017). Most drugs targeting class C GPCRs interact with allosteric binding sites that are topo- graphically distinct from orthosteric sites, allowing for better spatiotemporal control over receptor function and greater subtype selectivity (Conn et al., 2014). Allosteric modulators that enhance or inhibit orthosteric ligand binding and/or efficacy, a property known as cooperativity, are defined as positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs), respectively. Neutral allosteric ligands (NALs) bind to allosteric sites but display no cooperativity with orthosteric ligands. NALs are an interesting and important class of allosteric ligands as, although they may appear to display no This research was supported by the National Health and Medical Research Council of Australia [APP1084775, APP1123722, APP1085143]. K.J.G. and K.L. are recipients of Australian Research Council Future Fellowships [FT160100075, FT170100392]. https://.doi.org/10.1124/mol.117.111518. s This article has supplemental material available at molpharm. aspetjournals.org. ABBREVIATIONS: AC265347, 1-(1,3-benzothiazol-2-yl)-1-(2,4-dimethylphenyl)ethanol; AZ12216052, 2-[[(4-bromophenyl)methyl]thio]-N-[4-(1- methylpropyl)phenyl]acetamide; BINA, 39-[[(2-Cyclopentyl-2,3-dihydro-6,7-dimethyl-1-oxo-1H-inden-5-yl)oxy]methyl]-[1,19-biphenyl]-4-carboxylic acid; CaSR, calcium-sensing receptor; CCh, carbachol; CGP7930, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-b,b-dimethylbenzenepropanol; CPCCOEt, 7-(hydroxyimino)cyclopropa[b] chromen-1acarboxylate ethyl ester; DHPG, (S)-3,5-dihydroxyphenylglycine; DMEM, Dulbecco’s modified Eagle’s medium; EC 20 , 20% effective concentration; EC 80 , 80% effective concentration; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; GS39783, N4,N6-dicyclopentyl-2-(methylthio)-5-nitro-4,6-pyrimidinediamine; HBSS, Hank’s balanced salt solution; HEK293A, human embryonic kidney 293; iCa 21 , intracellular calcium; IP 1 , inositol 1-phosphate; mGlu, metabotropic glutamate receptor; ML 337, [2-fluoro-4-[2-(4-methoxyphenyl)- ethynyl]phenyl] [(3R)-3-hydroxy-1-piperidinyl]methanone; 5MPEP, 5-methyl-6-(phenylethynyl)-pyridine; MPEP, 2-methyl-6-(phenylethynyl)pyridine; NAL, neutral allosteric ligand; NAM, negative allosteric modulator; NPS2143, 2-chloro-6-[(2R)-3-([1,1-dimethyl-2-(2-naphthalenyl) ethyl]amino)-2-hydroxypropoxy]- benzonitrile; PAM, positive allosteric modulator; PHCCC, (E)-1,1a,7,7a-tetrahydro-7-(hydroxyimino)-N-phenylcyclopropa[b]chromene-1a-carboxamide; SAR, structure-activity relationship; 7TM, 7 transmembrane domain; VU0424465, (R)-5-((3-fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide; VU0483605, 3-chloro-N-[3-chloro-4-(4-chloro-1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)phenyl]-2-pyridinecarboxamide. 504 http://molpharm.aspetjournals.org/content/suppl/2018/03/07/mol.117.111518.DC1 Supplemental material to this article can be found at: at ASPET Journals on May 22, 2020 molpharm.aspetjournals.org Downloaded from